Many blood-ingesting pests are known to feed on humans and animals, and many pests are vectors for pathogenic microorganisms which threaten human and animal health, including commercially important livestock, pets and other animals. Various species of mosquitoes, for example, transmit diseases caused by viruses, and many are vectors for disease-causing nematodes and protozoa. Mosquitoes of the genus Anopheles transmit Plasmodium, the protozoan which causes malaria, a devastating disease which results in approximately 1 million deaths annually. The mosquito species Aedes aegypti transmits an arbovirus that causes yellow fever in humans. Other arboviruses transmitted by Aedes species include the causative agents of dengue fever, eastern and western encephalitis, Venezuelan equine encephalitis, St. Louis encephalitis, chikungunya, oroponehe and bunyarnidera. The genus Culex, which includes the common house mosquito C. pipiens, is implicated in the transmission of various forms of encephalitis and filarial worms. The common house mosquito also transmits Wuchereria bancrofti and Brugia malayi, which cause various forms of lymphatic filariasis, including elephantiasis. Trypanasoma cruzi, the causative agent of Chagas"" disease, is transmitted by various species of blood-ingesting Triatominae bugs. The tsetse fly (Glossina spp.) transmits African trypanosomal diseases of humans and cattle. Many other diseases are transmitted by various blood-ingesting pest species. The order Diptera contains a large number of blood-ingesting and disease-bearing pests, including, for example, mosquitoes, black flies, no-see-ums (punkies), horse flies, deer flies and tsetse flies.
Various pesticides have been employed in efforts to control or eradicate populations of disease-bearing pests, such as disease-bearing blood-ingesting pests. For example, DDT, a chlorinated hydrocarbon, has been used in attempts to eradicate malaria-bearing mosquitoes throughout the world. Other examples of chlorinated hydrocarbons are BHC, lindane, chlorobenzilate, methoxychlor, and the cyclodienes (e.g., aldrin, dieldrin, chlordane, heptachlor, and endrin). The long-term stability of many of these pesticides and their tendency to bioaccumulate render them particularly dangerous to many non-pest organisms.
Another common class of pesticides is the organophosphates, which is perhaps the largest and most versatile class of pesticides. Organophosphates include, for example, parathion, Malathion(trademark), diazinon, naled, methyl parathion, and dichlorvos. Organophosphates are generally much more toxic than the chlorinated hydrocarbons. Their pesticidal effect results from their ability to inhibit the enzyme cholinesterase, an essential enzyme in the functioning of the insect nervous system. However, they also have toxic effects on many animals, including humans.
The carbamates, a relatively new group of pesticides, include such compounds as carbamyl, methomyl, and carbofuran. These compounds are rapidly detoxified and eliminated from animal tissues. Their toxicity is thought to involve a mechanism similar to the mechanism of the organophosphates; consequently, they exhibit similar shortcomings, including animal toxicity.
A major problem in pest control results from the capability of many species to develop pesticide resistance. Resistance results from the selection of naturally-occurring mutants possessing biochemical, physiological or behavioristic factors that enable the pests to tolerate the pesticide. Species of Anopheles mosquitoes, for example, have been known to develop resistance to DDT and dieldrin. DDT substitutes, such as Malathion(trademark), propoxur and fenitrothion are available; however, the cost of these substitutes is much greater than the cost of DDT.
There is clearly a longstanding need in the art for pesticidal compounds that are pest- specific, that reduce or eliminate direct and/or indirect threats to human health posed by presently available pesticides, that are environmentally compatible in the sense that they are biodegradable, and are not toxic to non-pest organisms, and have reduced or no tendency to bioaccummulate.
Many pests, including for example blood-inbibing pests, must consume and digest a proteinaceous meal to acquire sufficient essential amino acids for growth, development and the production of mature eggs. Adult pests, such as adult mosquitoes, need these essential amino acids for the production of vitellogenins by the fat body. These vitellogenins are precursors to yolk proteins which are critical components of oogenesis. Many pests, such as house flies and mosquitoes, produce oostatic hormones that inhibit egg development by inhibiting digestion of the protein meal, and thereby limiting the availability of the essential amino acids necessary for egg development.
Serine esterases such as trypsin and trypsin-like enzymes (collectively referred to herein as xe2x80x9cTTLExe2x80x9d) are important components of the digestion of proteins by insects. In the mosquito, Aedes aegypti, an early trypsin that is found in the midgut of newly emerged females is replaced, following the blood meal, by a late trypsin. A female mosquito typically weighs about 2 mg and produces 4 to 6 xcexcg of trypsin within several hours after a ingesting blood meal. Continuous boisynthesis at this rate would exhaust the available metabolic energy of a female mosquito; as a result, the mosquito would be unable to produce mature eggs, or even to find an oviposition site. To conserve metabolic energy, the mosquito regulates TTLE biosynthesis with a peptide hormone named Trypsin Modulating Oostatic Factor (TMOF). Mosquitoes produce TMOF in the follicular epithelium of the ovary 12-35 hours after a blood meal; TMOF is then released into the hemolymph where it binds to a specific receptor on the midgut epithelial cells, signaling the termination of TTLE biosynthesis.
This regulatory mechanism is not unique for mosquitoes; flesh flies, fleas, sand flies, house flies, dog flies and other pests which ingest protein as part of their diet have similar regulatory mechanisms.
In 1985, Borovsky purified an oostatic hormone 7,000-fold and disclosed that injection of a hormone preparation into the body cavity of blood imbibed mosquitoes caused inhibition of egg development and sterility (Borovsky, D. [1985] Arch. Insect Biochem. Physiol. 2:333-349). Following these observations, Borovsky (Borovsky, D. [1988] Arch. Ins. Biochem. Physiol. 7:187-210) reported that injection or passage of a peptide hormone preparation into mosquitoes inhibited the TTLE biosynthesis in the epithelial cells of the gut. This inhibition caused inefficient digestion of the blood meal and a reduction in the availability of essential amino acids translocated by the hemolymph, resulting in arrested egg development in the treated insect. Borovsky observed that this inhibition of egg development does not occur when the oostatic hormone peptides are inside the lumen of the gut or other parts of the digestive system (Borovsky, D. [1988], supra).
Following the 1985 report, the isolated hormone, (a ten amino acid peptide) and two TMOF analogues were disclosed in U.S. Pat. Nos. 5,011,909 and 5,130,253, and in a 1990 publication (Borovsky, et al. [1990] FASEBJ 4:3015-3020). Additionally, U.S. Pat. No. 5,358,934 discloses truncated forms of the full length TMOF which have prolines removed from the carboxy terminus, including the peptides YDPAP (SEQ ID NO. 14), YDPAPP (SEQ ID NO. 15), YDPAPPP (SEQ ID NO. 16), and YDPAPPPP (SEQ ID NO. 17).
Neuropeptides Y (NPY) are an abundant family of peptides that are widely distributed in the central nervous system of vertebrates. NPY peptides have also recently been isolated and identified in a cestode, a turbellarian, and in terrestrial and marine molluscs (Maule et al., 1991 xe2x80x9cNeuropeptide F: A Novel Parasitic Flatworm Regulatory Peptide from Moniezia expansa (Cestoda: Cyclophylidea)xe2x80x9d Parasitology 102:309-316; Curry et al., 1992 xe2x80x9cNeuropeptide F: Primary Structure from the Turbellarian, Arthioposthia triangulataxe2x80x9d Comp. Biochem. Physiol. 101C:269-274; Leung et al., 1992 xe2x80x9cThe Primary Structure of Neuropeptide F (NPF) from the Garden Snail, Helix aspersaxe2x80x9d Regul. Pep. 41:71-81; Rajpara et al., 1992 xe2x80x9cIdentification and Molecular Cloning of Neuropeptide Y Homolog that Produces Prolonged Inhibition in Aplysia Neuronsxe2x80x9d Neuron. 9:505-513).
Invertebrate NPYs are highly homologous to vertebrate NPYs. The major difference between vertebrate and invertebrate NPYs occurs at the C-terminus where the vertebrate NPY has an amidated tyrosine (Y) whereas invertebrates have an amidated phenylalanine (F). Because of this difference, the invertebrate peptides are referred to as NPF peptides.
Cytoimmunochemical analyses of NPY peptides suggest that they are concentrated in the brain of various insects, including the Colorado potato beetle Leptinotarsa decemlineata (Verhaert et al., 1985 xe2x80x9cDistinct Localization of FMRFamide- and Bovine Pancreatic Polypeptide-Like Material in the Brain, Retrocerebal Complex and Subesophageal Ganglion of the Cockroach Periplaneta americanaxe2x80x9d L. Brain Res. 348:331-338; Veenstra et al., 1985 xe2x80x9cImmunocytochemical Localization of Peptidergic Neurons and Neurosecretory Cells in the Neuro-Endocrine System of the Colorado Potato Beetle with Antisera to Vertebrate Regulatory Peptidesxe2x80x9d Histochemistry 82:9-18). Partial purification of NPY peptides in insects suggests that both NPY and NPF are synthesized in insects (Duve et al., 1981 xe2x80x9cIsolation and Partial Characterization of Pancreatic Polypeptide-like Material in the Brain of the Blowfly alliphora vomitoriaxe2x80x9d Biochem. J. 197, 767-770).
Researchers have recently isolated two neuropeptides with NPF-like immunoreactivity from brain extracts of the Colorado potato beetle. The researchers purified the peptides using C18 reversed phase high pressure liquid chromatography (HPLC), and determined their structure using mass spectrometry. The deduced structures of these peptides are: Ala-Arg-Gly-Pro-Gln-Leu-Arg-Leu-Arg-Phe-amide (SEQ ID NO. 1) and Ala-Pro-Ser-Leu-Arg-Leu-Arg-Phe-amide (SEQ ID NO. 2) designated NPF I and NPF II, respectively (Spittaels, Kurt et al. [1996] xe2x80x9cInsect Neuropeptide F (NPF)-Related Peptides: Isolation from Colorado Potato Beetle (Leptinotarsa decemlineata) Brain,xe2x80x9d Insect Biochem. Molec. Biol. 26(4):375-382).
The subject invention provides materials and methods useful for the control of pests. In a specific embodiment the methods of the subject invention can be used for treating mosquito larvae to control mosquito populations. Specifically exemplified are recombinant hosts transformed to comprise and/or produce biological control agents capable of increasing the mortality of pests, including mosquitoes and mosquito larvae.
One aspect of the subject invention pertains to a composition comprising a host edible by mosquito larvae, wherein the cells of the host comprise a biological control agent that increases the mortality of the mosquito larvae. In a specific embodiment, the biological control agent inhibits biosynthesis of digestive enzymes, such as TTLE, thereby inhibiting food digestion. This inhibition of food digestion ultimately results in starvation and death of the mosquito larvae.
In a preferred aspect of the subject invention, an appropriate host is transformed with a polynucleotide encoding a polypeptide which acts to inhibit TTLE biosynthesis. Appropriate hosts include, but are not limited to, prokaryotic and eukaryotic cells, edible by pests including mosquito larvae. The biological control agents useful according to the subject invention include, but are not limited to, TMOF or functional equivalents thereof, NPF or functional equivalents thereof, and other agents identifiable by, for example, assays employing a TMOF receptor.
In a specific embodiment of the subject invention, salt water Chlorella, such as Chlorella desiccata are stably transformed with a plasmid carrying DNA encoding TMOF.
One embodiment of the present invention concerns a pesticide composition comprising a peptide having the formula:
A1A2A3A4A5Fl (Formula I) (SEQ ID NO. 8)
wherein:
A1 is selected from the group consisting of A, D, F, G, M, P, S and Y;
A2 is selected from the group consisting of A, D, E, F, G, N, P, S and Y;
A3 is selected from the group consisting of A, D, F, G, L, P, S and Y;
A4 is optionally present when A3 is present and is selected from the group consisting of A, F, G, L and Y;
A5 is optionally present when A4 is present and is selected from the group consisting of A, F, L and P;
Fl is a flanking region which is optionally present and is selected from the group consisting of: P, PP, PPP, PPPP (SEQ ID NO. 9), and PPPPP (SEQ ID NO. 10).
Preferably, the peptide does not comprise YDPAP6 (SEQ ID NO. 11), DYPAP6 (SEQ ID NO. 12), PAP6 (SEQ ID NO. 13), YDPAP (SEQ ID NO. 14), YDPAP2 (SEQ ID NO. 15), YDPAP3 (SEQ ID NO. 16), YDPAP4 (SEQ ID NO. 17), NPTNLH (SEQ ID NO. 18) or DF-OMe.
In a more specific aspect the peptide or protein comprises an amino acid sequence which consists essentially of the amino acid sequence of Formula 1. In a preferred aspect, the amino acid sequence is a TMOF or NPF fragment and lacks TMOF or NPF amino acids adjacent to the amino acid sequence of Formula I. Preferably the fragment has from 2-5 amino acids of TMOF. In still another aspect, the peptide consists of the amino acid sequence of Formula I.
In various embodiments, either A3A4A5, A3A4A5Fl, A4A5,Fl, A5 or A5Fl are not present. Where A5 is not present, Fl may be attached directly to A4. Where A4A5 is not present, Fl may be attached directly to A3. Finally, where A3A4A5 is not present, Fl may be attached directly to A2.
Preferred peptides are selected from the group consisting of: AAP (SEQ ID NO. 19), ADP (SEQ ID NO. 20), ADPAP (SEQ ID NO. 21), APA (SEQ ID NO. 22), DAA (SEQ ID NO. 23), DF (SEQ ID NO. 24), DPA (SEQ ID NO. 25), DY (SEQ ID NO. 26), DYP (SEQ ID NO. 27), FAP (SEQ ID NO. 28), FDP (SEQ ID NO. 29), FDPAP (SEQ ID NO. 30), FSP (SEQ ID NO. 31), MPDYP5 (SEQ ID NO. 32), PAA (SEQ ID NO. 33), PAP (SEQ ID NO. 34), Y(D)DP (SEQ ID NO. 35), Y(D)DPAP (SEQ ID NO. 36), YAP (SEQ ID NO. 37), YD (SEQ ID NO. 38), YDA (SEQ ID NO. 39), YDAAP (SEQ ID NO. 40), YDF (SEQ ID NO. 41), YDFAP (SEQ ID NO. 42), YDG (SEQ ID NO. 43), YDLAP (SEQ ID NO. 44), YDP (SEQ ID NO. 45), (D)YDP (SEQ ID NO. 46), YDPAF (SEQ ID NO. 47), YDPAL (SEQ ID NO. 48), (D)YDPAP (SEQ ID NO. 49), YDPFP (SEQ ID NO. 50), YDPGP (SEQ ID NO. 51), YDPLP (SEQ ID NO. 52), YEPAP (SEQ ID NO. 53), YFPAP (SEQ ID NO. 54), YNPAP (SEQ ID NO. 55) and YSF (SEQ ID NO. 56).
A further embodiment of the present invention comprises a peptide having the formula
A1A2 (Formula II) (SEQ ID NO. 62)
wherein
A1 is an amino acid selected from the group consisting of A, D, F, M, and Y, and
A2 is an amino acid selected from the group consisting of A, D, E, P, and Y.
In a preferred embodiment, the subject invention is directed to peptides of Formula II wherein A1 and A2 are independently selected from the group consisting of A, D, and Y.
Specifically exemplified as another embodiment are methods using an NPF peptide having the sequence Ala-Arg-Gly-Pro-Gln-Leu-Arg-Leu-Arg-Phe-amide (SEQ ID NO. 1) or Ala-Pro-Ser-Leu-Arg-Leu-Arg-Phe-amide (SEQ ID NO. 2).
The term xe2x80x9cpesticidal polypeptidexe2x80x9d is used herein to indicate NPF and TMOF peptides, as well as fragments, derivatives and analogues and functional equivalents of NPF and TMOF. The present invention also provides analogues of the pesticidal polypeptides which have one or more amino acid substitutions forming a branched peptide (e.g., by substitution with an amino acid or amino acid analogue having a free amino- or carboxy-side chain that forms a peptide bond with a sequence of one or more amino acids, including but not limited to prolines) or allowing circularization of the peptide (e.g., substitution with a cysteine, or insertion of a cysteine at the amino- or carboxy-terminus or internally, to provide a sulfhydryl group for disulfide bond formation).
The pesticidal polypeptides of the present invention are particularly advantageous because their smaller size permits more rapid and efficient penetration into the midgut. In addition, they are less expensive to produce by conventional chemical methods.
In one embodiment, the subject invention provides pesticidal polypeptides having a C-terminus arginine. In a preferred embodiment, these short polypeptides can be joined to form polymers of repeating units. Specifically exemplified herein is the (DPAR)4 (SEQ ID NO. 61) polymer which can be broken into four DPAR (SEQ ID NO. 60) units in the gut of the pest. Advantageously, the short pesticidal polypeptides connected by arginine (or other readily cleavable residue) can penetrate the midgut of the pest efficiently.
Also included in this invention are addition salts, complexes, or prodrugs such as esters of the pesticidal polypeptide, especially the nontoxic pharmaceutically or agriculturally acceptable acid addition salts. The acid addition salts can be prepared in standard manner in a suitable solvent from the parent compound and an excess of an acid, such as hydrochloric, hydrobromic, sulfuric, phosphoric, acetic, maleic, succinic, ethanedisulfonic or methanesulfonic acids. Also, the N-terminus and C-terminus of the pesticidal polypeptides can be chemically modified to further inhibit proteolysis by metabolic enzymes.
The analogues of the pesticidal polypeptides of the present invention also include polypeptides having NPF and/or TMOF amino acid sequences in which one or more of the amino acid residues has been substituted by an amino acid in the D- conformation. The presence of D-conformation amino acids can inhibit the ability of proteases to degrade the peptides of the subject invention. Polypeptides having the above sequences in which only conservative substitutions have been made are also provided by the present invention.
Also, derivation of the pesticidal polypeptides with long chain hydrocarbons will facilitate passage through the cuticle into the pest body cavity. Accordingly, a further embodiment of the subject invention pertains to compositions comprising the pesticidal polypeptides bound to lipids or other carriers.
Yet another aspect of the subject invention pertains to polynucleotides encoding the pesticidal polypeptides of the subject invention. These polynucleotides can readily be synthesized by a person skilled in the art. The sequences may be used to transform an appropriate host to confer upon that host the ability to express the novel peptides. Hosts of particular interest include bacteria, algae, yeasts, and plants. Viruses may also be modified to comprise polynucleotide sequences encoding the pesticidal polypeptides of the present invention. For each of these hosts, the polynucleotides may be specifically designed by a person skilled in the art to utilize codons known to be optimally expressed in the particular hosts. Advantageous promoters are also readily incorporated into the polynucleotides. Bacteria, yeasts, plants, algae, viruses, and other hosts each may be used to produce peptides for further use, or these hosts can be used as vehicles for direct application of the peptide to the target pest. Plants can be transformed to make the plant toxic to a target pest species which feeds on that plant. Methods for transforming plant cells utilizing, for example, Agrobacteria are well known to those skilled in the art.
As used herein, the term xe2x80x9cpesticidally effectivexe2x80x9d is used to indicate an amount or concentration of a pesticide which is sufficient to reduce the number of pests in a geographical area, as compared to a corresponding geographical area in the absence of the amount or concentration of the pesticide.
The term xe2x80x9cpesticidalxe2x80x9d is not intended to refer only to the ability to kill pests, but also includes the ability to interfere with a pest""s life cycle in any way that results in an overall reduction in the pest population. For example, the term xe2x80x9cpesticidalxe2x80x9d included inhibition or elimination of reproductive ability of a pest, as well as inhibition of a pest from progressing from one form to a more mature form, e.g., transition between various larval instars or transition from larvae to pupa or pupa to adult. Further, the term xe2x80x9cpesticidalxe2x80x9d is intended to include all phases of a pest life cycle; thus, for example, the term includes larvicidal, ovicidal, and adulticidal action.
The word xe2x80x9ctransformxe2x80x9d is broadly used herein to refer to introduction of an exogenous polynucleotide sequence into a prokaryotic or eukaryotic cell by any means known in the art (including, for example, direct transmission of a polynucleotide sequence from a cell or virus particle as well as transmission by infective virus particles and transmission by any other known means for introducing a polynucleotide into a cell), resulting in a permanent or temporary alteration of genotype and in an immortal or non-immortal cell line.
The terms xe2x80x9cpeptide,xe2x80x9d xe2x80x9cpolypeptide,xe2x80x9d and xe2x80x9cproteinxe2x80x9d as used herein are intended to refer to amino acid sequences of any length.
Another aspect of the subject invention pertains to a method of controlling pests comprising administering to said pest an effective amount of a peptide of the subject invention.
The subject invention provides pesticidal compositions wherein the pesticidal polypeptides are formulated for application to the target pests, or their situs. In a specific embodiment, the present invention provides recombinant hosts which express a polynucleotide encoding a pesticidal polypeptide to produce the pesticidal polypeptide. The recombinant host may be, for example, prokaryotic or eukaryotic. In a specific example, yeast or algae are transformed to express a pesticidal polypeptide of the subject invention. The transformed hosts are then applied to water areas where mosquito larvae will ingest the transformed host resulting in control of the mosquitoes by the pest control agent.
Preferably, the subject peptides have an LD50 against mosquito larvae of less than 3.0 xcexcmoles/ml. More preferably, the peptides have an LD50 of less than 2.0 xcexcmoles/ml, and, most preferably, the peptides have an LD50 of less than 1.0 xcexcmoles/ml. As used herein, xe2x80x9cLD50xe2x80x9d refers to a lethal dose of a peptide able to cause 50% mortality of larvae maintained on ad iet of 1 mg/ml autoclaved yeast (Borovsky and Mahmood [1995] xe2x80x9cFeeding the mosquito Aedes aegypti with TMOF and its analogs; effect on trypsin biosynthesis and egg development,xe2x80x9d Regulatory Peptides 57:273-281).
Another aspect of the subject invention pertains to methods of controlling pests comprising preparing a host to produce a pesticidal polypeptide wherein the host is edible by the target pest and administering the host to the target pest.
Another aspect of the subject invention pertains to pesticidal polypeptides and other pesticidal compounds used in conjunction with a marker to aid in the administration and/or monitoring of the biological control agent. Such markers include, for example, Green Fluorescent Protein (GFP). This protein fluoresces and provides a means to determine whether target pests, such as mosquitoes, are eating or have been treated by the biological control agent. Target pests which have eaten the biological control agent fused with the GFP will fluoresce. This fluorescence can be employed as an analytical measurement which indicates whether target pests such as mosquito larvae are indeed consuming the pesticidal polypeptide or other pesticidal compound Such measurements are useful for determining the amount of pesticidal polypeptide which must be applied to maintain a pesticidally effective amount of the pesticidal polypeptide.